WO2013036819A1 - Procédés d'augmentation du nombre de cellules cibles récupérées d'un échantillon fluide - Google Patents

Procédés d'augmentation du nombre de cellules cibles récupérées d'un échantillon fluide Download PDF

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Publication number
WO2013036819A1
WO2013036819A1 PCT/US2012/054241 US2012054241W WO2013036819A1 WO 2013036819 A1 WO2013036819 A1 WO 2013036819A1 US 2012054241 W US2012054241 W US 2012054241W WO 2013036819 A1 WO2013036819 A1 WO 2013036819A1
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WO
WIPO (PCT)
Prior art keywords
filter
cells
target cells
animal
target
Prior art date
Application number
PCT/US2012/054241
Other languages
English (en)
Inventor
Robert J. DISTEL
Yvon Cayre
Original Assignee
Dana Farber Cancer Institute, Inc.
Screencell
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dana Farber Cancer Institute, Inc., Screencell filed Critical Dana Farber Cancer Institute, Inc.
Priority to CN201280054752.5A priority Critical patent/CN103917871A/zh
Priority to CA2847891A priority patent/CA2847891A1/fr
Priority to US14/342,888 priority patent/US20150110717A1/en
Priority to EP12766511.5A priority patent/EP2753926A1/fr
Priority to JP2014529908A priority patent/JP2014526251A/ja
Publication of WO2013036819A1 publication Critical patent/WO2013036819A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • This invention relates to methods and materials for increasing the number of target cells recovered from a fluid sample containing cells, and more particularly to increasing the number of target cells recovered from a fluid sample containing cells by isolating the target cells on a filter and then implanting the filter containing the target cells in an immunodeficient non-human animal, where at least some of the target cells can proliferate.
  • CTCs circulating tumor cells
  • target cells can be retained on or in a filter on the basis of size, and then the filter containing the target cells can be implanted into a non-human animal, preferably an immunodeficient non-human animal, where the cells can proliferate.
  • a non-human animal preferably an immunodeficient non-human animal
  • Such methods can be used to increase the number of rare circulating cells such as CTCs recovered from a peripheral blood sample and create individualized animal models of a patient's tumor that can be used, for example, to evaluate the patient's tumor, assess metastatic potential of the cells, and to determine responsiveness of the cells to different chemotherapeutics .
  • this document features a method of increasing the number of target cells from a fluid sample comprising cells.
  • the method includes providing a filter comprising one or more target cells, the one or more target cells obtained from a fluid, target cell and non-target cell containing sample by passage of the sample through a filtration device comprising the filter, wherein the size of pores in the filter causes the target cells to be retained on or in the filter; and implanting the filter and the one or more target cells on the filter in a non-human animal, e.g., an
  • immunodeficient non-human animal such as an immunodeficient mouse
  • immunodeficient non-human animal such as an immunodeficient mouse
  • the immunodeficient mouse can be homozygous for the severe combined immune deficiency (SCID) spontaneous mutation (Prkdc scld ); homozygous for the nude spontaneous mutation (Foxnl nu/nu ); homozygous for a Ragl mutation; homozygous for a Rag2 mutation; or homozygous for both the Ragl and the Rag2 mutation.
  • SCID severe combined immune deficiency
  • Prkdc scld homozygous for the nude spontaneous mutation
  • Roxnl nu/nu homozygous for a Ragl mutation
  • homozygous for a Rag2 mutation homozygous for a Rag2 mutation
  • homozygous for both the Ragl and the Rag2 mutation or homozygous for both the Ragl and the Rag2 mutation.
  • the method further can include providing one to four additional filters (e.g., one, two, three or four additional filters), each additional filter comprising one or more target cells, and implanting the first and additional filters in the immunodeficient non-human animal.
  • the first and additional filters can be obtained from a single filtration device or from separate filtration devices.
  • the method further can include, before implanting the filter and the one or more target cells on the filter, stacking the filters substantially on top of each other to produce a multi-layered culture device.
  • any of the methods described herein further can include, before implanting the filter and the one or more target cells on the filter, contacting the surface of the filter and any additional filters comprising the target cells with a composition that can transition from a liquid to gel phase without lethal or toxic effects on the target cells.
  • the composition can include one or more extracellular matrix (ECM) components (e.g., reconstituted basement membrane).
  • ECM extracellular matrix
  • the filter can include one or more compounds immobilized thereto.
  • one or more compounds can be administered to the immunodeficient animal.
  • the one or more compounds can be selected from the group consisting of a growth factor, an extracellular matrix protein, an enzyme, a reporter molecule, a liposome, and a nucleic acid.
  • the growth factor can be epidermal growth factor (EGF), platelet derived growth factor (PDGF), keratinocyte growth factor (KGF), a fibroblast growth factor (FGF), or a transforming growth factor (TGF).
  • the extracellular matrix protein can be collagen, laminin, fibronectin, or heparan sulfate.
  • the reporter molecule can include a fluorophore-quencher dual labeled probe that is a substrate for a metalloproteinase.
  • the method further can include monitoring growth of the cells in the immunodeficient animal.
  • the fluid, cell-containing sample can include peripheral blood cells or can include cells from urine, bone marrow, lymph, lymph node, spleen, cerebral spinal fluid, ductal fluid, a biopsy specimen, or a needle biopsy aspirate.
  • the method further can include, before the implanting step, culturing the one or more target cells.
  • the target cells can be cancer cells, circulating cancer cells, fetal cells, or stem cells (e.g., endothelial stem cells or mesenchymal stem cells).
  • stem cells e.g., endothelial stem cells or mesenchymal stem cells.
  • this document features a non-human immunodeficient animal that includes at least one implanted filter, the filter comprising a plurality of target cells obtained from a fluid, target cell and non-target cell containing sample by passage through a filtration device comprising the filter, wherein the size of pores in the filter causes the target cells to be retained on or in the filter.
  • the animal further can include one to four additional implanted filters (e.g., one, two, three or four), each said additional filter comprising one or more target cells.
  • the first and additional filters can be obtained from a single filtration device or from separate filtration devices.
  • the first and additional filters can be substantially stacked on top of each other to produce a multi-layered three-dimensional culture device.
  • the surface of the filter and any additional filters comprising the target cells can include a composition that can transition from a liquid to gel phase without lethal or toxic effects on the target cells (e.g., human target cells).
  • This document also features a method of testing for the presence of tumor cells in fluid sample comprising test cells.
  • the method includes providing a filter comprising a plurality of test cells obtained from a fluid, test cell-containing sample by passage through a filtration device comprising the filter, wherein the size of pores in the filter causes one or more of the test cells to be retained on or in the filter;
  • test cells can be human cells.
  • the fluid, cell-containing sample can include peripheral blood cells or comprise cells from urine, bone marrow, lymph, lymph node, spleen, cerebral spinal fluid, ductal fluid, a biopsy specimen, or a needle biopsy aspirate.
  • the method further can include administering a chemotherapeutic agent to the non-human animal if the tumor is present; and monitoring the tumor for responsiveness to the chemotherapeutic agent.
  • this document features a method of increasing the number of target cells from a fluid sample comprising cells.
  • the method includes providing a fluid, target cell- and non-target cell-containing sample; passing the sample through a filtration device, the device comprising a filter support fastened to a filter, a compartment having an upper opening and a lower opening, and means mobile relative to the compartment for applying a force to the support and releasing the support; removing, from the device, the filter containing one or more target cells; and implanting the filter and the target cells on or in the filter into the immunodeficient non-human animal, wherein some or all of the one or more target cells on or in the implanted filter proliferate in the immunodeficient animal.
  • FIG. 1 is a perspective view of a ScreenCell® filtration device for recovering target cells from a fluid sample according to an embodiment described herein.
  • FIGs. 2A and 2C are perpendicular axial sections of the embodiment of FIG. 1, with the parts illustrated in FIG. 1 assembled.
  • the circled portions in FIGs. 2A and 2C are presented in enlarged view in FIGs. 2B and 2D, respectively.
  • FIGs. 3A- 30 are elevation or cross-section views of the ScreenCell® filtration device of FIG. 1 from storage configuration (3 A) through each step of using the device (3B-30) for isolating and recovering target cells from a fluid sample.
  • FIG. 4 is perspective view of one embodiment of a ScreenCell® filtration device, before use.
  • FIG. 5 diagrammatically represents the embodiment of the ScreenCell® device illustrated in FIG. 4 after removal of a protective film and before insertion of a vacuum tube.
  • FIG. 6 diagrammatically represents the embodiment of the ScreenCell® device illustrated in FIGs. 4 and 5, after insertion of the vacuum tube,
  • FIG. 7 diagrammatically represents the embodiment of the ScreenCell® device illustrated in FIGS. 4-6 during removal of the vacuum tube and a protective cylinder.
  • FIG. 8A is a photomicrograph of live H2030 cells following filtration through a ScreenCell® Cyto device.
  • FIG. 8B is a photomicrograph of the cells from FIG. 8A after culturing the filter for 4 days in culture medium.
  • Cells in FIGs. 8 A and 8B are representative of 8 independent experiments; cells were observed under a microscope (x 40).
  • Like reference symbols in the various drawings indicate like elements.
  • fluid sample containing cells refers to a liquid containing a suspension of cells.
  • biological fluids such as blood (e.g., peripheral blood or umbilical cord blood), urine, lymph, cerebral spinal fluid, or ductal fluid, or such fluids diluted in a physiological solution (e.g., saline, phosphate- buffered saline (PBS), or tissue culture medium), or cells obtained from biological fluids (e.g., by centrifugation) and suspended in a physiological solution.
  • physiological solution e.g., saline, phosphate- buffered saline (PBS), or tissue culture medium
  • cells obtained from biological fluids e.g., by centrifugation
  • Other examples of a "fluid sample containing cells” include cell suspensions (in
  • physiological solutions obtained from bone marrow aspirates, needle biopsy aspirates or biopsy specimens from, for example, lymph node or spleen.
  • fluid samples can be obtained from any mammalian subject, including humans, monkeys, mice, rats, rabbits, guinea pigs, dogs, or cats. Fluid samples from human subjects are particularly useful.
  • the red blood cells can be selectively lysed using, for example, a buffer containing ammonium chloride or saponin or removed by, for example, density gradient sedimentation or hetastarch aggregation.
  • viable target cells can be recovered from a fluid sample on or in a filter, which then can be implanted in an immunodeficient non-human animal, where the target cells can proliferate.
  • Target cells can include fetal blood cells, circulating tumor cells (CTCs), disseminated tumor cells (DTCs) (i.e., tumor cells in bone marrow), or stem cells (e.g., cancer stem cells, mesenchymal stem cells, or endothelial stem cells).
  • CTCs circulating tumor cells
  • DTCs disseminated tumor cells
  • stem cells e.g., cancer stem cells, mesenchymal stem cells, or endothelial stem cells.
  • fetal blood cells can be recovered from a sample of maternal blood (optionally diluted in a physiological solution) and used for non-invasive prenatal diagnosis.
  • One or more filters containing target cells recovered from a patient having, or suspected of having, a cancer can be implanted in the immunodeficient non-human mammal to increase the number of target cells (including the progeny of target cells trapped in or on a filter) for further characterization, including one or more of genomic, proteomic, immunocytochemistry, or fluorescence in situ hybridization (FISH) assays, to, for example, aid in prognosis determination.
  • Implantation into an immunodeficient animal also allows a determination of the capability of the target cells to initiate tumors and metastasize and/or a determination of the responsiveness of the cell to one or more chemotherapeutic agents.
  • Non-target cells are all the cells in a fluid sample containing cells other than the target cells.
  • non-target cells would include red blood cells, lymphocytes (T and B), monocytes, and granulocytes, which are smaller than most cancer cells.
  • the fluid sample containing cells is, for example, a cell suspension prepared from lymph node tissue and the target cells are cancer cells
  • non-target cells will include lymphocytes (T and B), monocytes, macrophages, and granulocytes, which are smaller than most cancer cells.
  • a fluid sample containing target and non-target cells can be passed through a filtration device that includes a filter, wherein the size of the pores in the filter causes the target cells to be retained on or in the filter.
  • a buffer containing culture medium e.g., RPMI such as RPMI 1640, DMEM, or MEM
  • bovine serum albumin e.g., bovine serum albumin
  • red blood cell lysis agent such as ammonium chloride, saponin, or potassium bicarbonate
  • a biocidal agent such as sodium azide or a hypochlorite solution (0.1 to 2 mM)
  • a calcium channel blocker such as amlodipine, benidipine, or barnidipine, and incubated for one to five minutes (e.g., one minute, two minutes, three minutes, four minutes, or five minutes).
  • the buffer can be supplemented with 0.2 to 2 g (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8. 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 g) of BSA, 0.01 to 0.1 g (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 1.0 g) of a red blood cell lysis agent, 0.1 to 2 mM (e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8.
  • BSA BSA
  • 0.01 to 0.1 g e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 1.0 g
  • 0.1 to 2 mM e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8.
  • a cell culture medium e.g., RPMI
  • RPMI cell culture medium
  • substantially all, i.e., greater than 99%, of the non-target cells pass through the filter.
  • the non-target cells of the fluid sample can pass through the filter by the application of a vacuum to the underside of the filter.
  • the non-target cells of the sample are caused to pass through the filter by introducing a tube inside the holder of the device which, once its rubber cap is pierced, establishes a pressure difference between the blood volume and the vacuum in the tube, forcing blood through the filter and into the tube.
  • the filtration device containing the filter is not limited to a particular structure and can be of any shape, size, or material as long as it is a) capable of receiving the fluid sample containing the cells and supporting a filter in which the size of its pores retains the target cells on or in the filter, and b) is configured for removal of the fluid after passage of the sample through the filter.
  • a filtration device can be composed of one or more of the following: a plastic made of one or more polymers such as polycarbonate, polyamide, polyvinyl chloride, polypropylene, polyethylene, or a polyetheretherketone such as PEEKTM; a metal alloy such as stainless steel (e.g., surgical steel); a ceramic, glass; or a composite material.
  • the filtration devices of ScreenCell® are particularly useful and have been used in the methods described herein. See also the filtration devices described in U.S. Patent Publication No. 201 10070642, U.S. Patent Publication No. 201 10104670, U.S. Patent Publication No. 20090081772, U. S. Provisional Application No. 61/417,526, WO201 1055091 , WO2009106760, and WO2009047436, each of which is incorporated by reference in its entirety.
  • the filtration device includes a compartment for receiving a fluid sample and a filter mounted, at least temporarily, onto an opening of the compartment.
  • the filtration device further can include a needle mounted, at least temporarily, onto the opening of the compartment.
  • the filter is located between the needle and the interior capacity of the compartment.
  • the mounted needle is designed to pierce the plug of a vacuum tube (e.g., a blood vacuum tube), creating negative pressure relative to ambient pressure in order to aspirate the liquid through the filter.
  • a vacuum tube e.g., a blood vacuum tube
  • a filtration device (ScreenCell®, Paris - France) that includes a reservoir or compartment, 102, an end-piece 104, a seal 106, a filter with its support 108, a movable means 110, a seal 112 and a plug 114.
  • the compartment 102 is substantially cylindrically shaped. Its upper end can be sealed by the plug 114 in such a way that it is impermeable.
  • the lower end of the compartment 102 has, on its external surface, discontinuous rings with gaps which guide legs on the movable means 1 10, said rings guiding the body of the movable means 1 10.
  • This movable means 1 10 is generally cylindrical in shape and is supplied with two legs extending towards the end-piece 104 and narrowing together in this direction so that there is a separation between them measuring less than the diameter of the filter support 108.
  • this particular shape notably that of the legs 116 curved towards each other, allows the movable means 1 10, after it is removed from the end-piece 104, to apply pressure to the filter support 108 so that the filter support is released from the compartment 102, along with its filter, while the movable means is being advanced towards the filter support 108.
  • the ends of the legs 116 of the movable means 1 10 and the lower end of the compartment 102 are designed to be inserted into a cell culture box or well.
  • the diameter of the discontinuous ring at the end of the compartment 102 is designed so that the compartment can be supported on the edge of a cell culture box or well.
  • An end-piece or adaptor 104 is located at the lower opening of the compartment 102. End piece or adaptor 104 grips the exterior wall of the
  • End-piece or adaptor 104 is detachable, impermeable, and sterile.
  • the lower narrow opening in the end-piece or adaptor 104 is sufficiently long to enable a leak-free mechanical fit of the aperture of a needle (see FIGs. 3B to 3D).
  • the end-piece 104 has rotation locking means for gripping the lugs in the manner known. In this way, the end-piece 104 allows the filter-holder to be held in place during filtration. In addition, the end-piece 104 protects the filter from splashes and any potential contamination.
  • the lower end of the compartment 102 When attached, the lower end of the compartment 102 has an opening which discharges onto the filter held by the filter support 108, which is itself held in position, on the one hand, by the lower end of the compartment 102 and, on the other, by the end-piece 104.
  • the filter support 108 is shaped as a ring-like disk.
  • the filter is micro-perforated and is fused to the underside of the filter support 108, then inserted along with it into the lower end of the compartment 102.
  • the filter support 108 can be ring-shaped and made, for example, of plastic such as polyvinyl chloride (PVC) or a metal alloy such as surgical steel.
  • a filter support made of surgical steel is particular useful in the filtration device.
  • the thickness of the ring is designed to allow it to be scanned.
  • the filter support can include an identifier such that the collected cells can be associated with a patient.
  • the external diameter of the filter support can be, for example, 12 to 13 mm such as 12.6 mm and the diameter of the filter to which the filter support 108 is attached can be 5.5 to 6.5 mm such as 5.9 mm.
  • the compartment 102, the end-piece 104 and the movable means 1 10 are composed of polypropylene, for example.
  • the seals 106 and 112 are made of silicone, for example.
  • FIGs. 2A and 2C are views of perpendicular axial sections of the embodiment depicted in FIG. 1.
  • FIGS. 2B and 2D are enlarged views of the circled portions of FIGs. 2A and 2C, respectively.
  • FIG. 3A represents the ScreenCell® filtration device in elevation, in its storage configuration.
  • FIG. 3B represents the insertion of the end-piece 104 into the aperture 181 of a needle 180, which has another very fine, beveled end 182 to make it easier to pierce a vacuum tube plug.
  • the aperture 181 of the needle 180 is
  • the end 182 of the needle 180 is preferentially metallic.
  • the needle 180 can be positioned on the end-piece 104 either before or after liquid (not shown), for example blood, is introduced into the compartment 102, through its upper opening.
  • FIG. 3C illustrates the vacuum tube 185 plug 186 beginning to be pierced, once the needle 180 is impermeably joined to the end-piece 104.
  • FIG. 3D illustrates the plug 186 completely pierced through by the needle 180, connecting the interior of the negative pressure vacuum tube 185, through the filter 108, to the volume of the compartment 102 holding the liquid containing the cells of interest.
  • the inner volume of the vacuum tube 185 is greater than the volume of the liquid to be filtered.
  • some target cells in the fluid sample present in the compartment 102 which are larger in diameter, are retained by the filter 108 while substantially all of the liquid contents and the cells smaller in dimension than the target cells are aspirated into the vacuum tube 185, through the filter 108.
  • the end-piece 104 is removed after being rotated to release it from the lugs 118. Then, as illustrated in FIGs. 3G and 3H, the end of the compartment 102 is inserted into a cell culture box or well 130.
  • the end near to the legs 1 16 of the movable means 110 and the lower end of the compartment 102 are designed to be inserted into a cell culture box or well.
  • the discontinuous ring at the end of the compartment 102 has a diameter allowing it to be supported on the edge of the culture box or well 130.
  • the movable means 110 can in this position still move parallel to the axis of the compartment 102.
  • the legs 1 16 of the mobile means when moved by the operator's fingers, apply vertical downward pressure on the filter support 108 and release it from the lower end of the
  • the compartment 102 and the mobile means 110 are removed from the cell culture box or well 130.
  • FIGs. 4-7 relate to particular embodiments of the ScreenCell® filtration device that uses a protective guiding cylinder for the vacuum tube.
  • FIGs. 4 to 7 show the compartment 102, the movable means 1 10 and a protective cylinder 502 attached to the compartment 102 by a two-part connection means, 504 and 520.
  • the protective cylinder includes the connection means part 504, a frosted part 506, a transparent part 508 and, on an opening opposite the compartment 102, a protective film 510.
  • Part 520 is formed inside the end of the compartment 102.
  • FIG. 7 shows a particular embodiment of part 520 comprising 4 prongs 522 laterally positioned on a cylindrical part in co-axial relation to the compartment 102.
  • part 504 is comprised of four grooves with profiles corresponding to those of the prongs. These grooves 524 extend in a elliptical fashion, from an opening designed to accommodate a prong 522 towards the interior of the protective cylinder 502 so that by rotating the protective cylinder 502 as indicated by an arrow in FIG. 7 causes each prong 522 to advance into the corresponding groove 524 and the protective cylinder 502 to be tightened onto the compartment 102.
  • the protective cylinder 502 is attached to the end-piece carrying the needle 180 as follows.
  • the needle 180 is embedded in the lower part of part 504, which is the part facing the compartment 502.
  • Part 504 is force-mounted onto part 506 by means of 4 spokes. The spokes are located on part 504 and are inserted into four grooves located on part 506.
  • Part 506 serves to isolate and protect the needle 180.
  • the transparent part 508 enables the user to verify the status and completion of filtration.
  • the protective film 510 which covers and seals the entire lower opening of the cylinder 502 is furnished with a lateral part extending a short distance from the cylinder 502 (illustrated in FIG. 4). This lateral part allows the film 510 to be easily removed.
  • the protective film 510 protects the user from access to the needle 180.
  • the film 510 also protects the needle 180 from the risk of clogging and/or contamination.
  • the cylinder 502 can be discreetly colored, for example blue, green or yellow, depending on the purposes for which the filtration device is used (cytological, molecular biology and culture studies, respectively).
  • a filter used in the methods described herein contains pores that cause the target cells to be retained on or in the filter.
  • a suitable filter can include between 50,000 and 200,000 pores/cm 2 (e.g., 75,000 to 150,000 pores/cm 2 , 90,000 to 115,000 pores/cm 2 , or 95,000 to 1 10,000 pores/cm 2 ) with an average diameter of 5.5 ⁇ to about 7.5 ⁇ . In one embodiment, the filter has approximately 100,000 pores/cm 2 with an average diameter of about 6.5 ⁇ .
  • the pore size used in any particular application will depend on the relative size of the target cells and non-target cells.
  • Target cells to be retained on a filter will generally have a diameter (or longest dimension) of >20 ⁇ and ⁇ 50 ⁇ . Naturally, at least most of the non-target cells in a fluid sample containing cells will have diameters (or largest dimensions) significantly smaller than that of the target cells in the fluid sample and smaller than the diameter (or largest dimension) of the pores in a filter of interest.
  • the filter can be composed of any biocompatible material, including, for example, a biocompatible polymer that is biodegradable or nonbiodegradable.
  • biocompatible polymers include, but are not limited to, poly(ester amide), polyhydroxyalkanoates (PHA), poly(3-hydroxyalkanoates) such as poly(3- hydroxypropanoate), poly(3-hydroxybutyrate), poly(3 -hydroxy valerate), poly(3- hydroxyhexanoate), poly(3-hydroxyheptanoate) and poly(3 -hydroxy octanoate), poly(4-hydroxyalkanaote) such as poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanote), poly(4-hydroxyheptanoate), poly(4-hydroxyoctanoate) and copolymers including any of the 3 -hydroxy alkanoate or 4-hydroxyalkanoate monomers described herein or blends thereof, poly(D,L-lactide), poly(L-lactide), polyglycolide, poly(D,L-lactide-co-glycolide), poly(L-l
  • the filter is a polycarbonate filter.
  • the filter is a polycarbonate track-edged filter from Whatman (Kent, UK), EMD Millipore (Billerica, Massachusetts), Membrane solutions (Piano, Texas), or it4ip (Seneffe, Belgium). Polycarbonate track-edged filters can be implanted in an immunosuppressed mouse with no adverse effects.
  • the surface of the filter can be modified by, for example, immobilization of one or more compounds or by treatment to render the surface more hydrophilic.
  • a growth factor e.g., a polycarbonate filter
  • growth factors include epidermal growth factor (EGF), platelet derived growth factor (PDGF), keratinocyte growth factor (KGF), a fibroblast growth factor (FGF), and a transforming growth factor (TGF).
  • growth factors include epidermal growth factor (EGF), platelet derived growth factor (PDGF), keratinocyte growth factor (KGF), a fibroblast growth factor (FGF), and a transforming growth factor (TGF).
  • extracellular matrix proteins include collagen, laminin, fibronectin, and heparan sulfate.
  • one or more reporter molecules can be immobilized on the filter such that cell growth can be detected during culture of the cells or after implantation in an immunodeficient non-human animal.
  • a reporter molecule can include a fluorophore-quencher dual labeled probe that is a substrate for a metalloproteinase (MMP).
  • MMP metalloproteinase
  • a substrate such as MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH, where MCA refers to methoxycoumarin and DPA refers to dinitrophenyl, can be used.
  • MCA metalloproteinase
  • Most MMPs associated with tumor growth can cleave such as substrate.
  • Fluorescent MCA is quenched by DPA until the peptide is cleaved by a MMP between the Gly and Leu residues. Detecting of fluorescent MCA is indicative of growth of the cells.
  • fluorophore and quencher molecules can be used.
  • the reporter molecule immobilized on the filter is an in vivo targeted, activatable optical imaging probe based on a fluorophore-quencher pair bound to a targeting ligand. See, Ogawa et ah, Mol. Pharm. 6(2): 386-395 (2009). With this system, fluorescence is quenched by the fluorophore-quencher interaction outside the target cells, but is activated within the target cells by dissociation of the fluorophore-quencher pair in lysosomes/endosomes.
  • the rhodamine core fluorophore TAMRA and QSY7 quencher pair are particularly useful for in vivo imaging.
  • Suitable target ligands include for example, a receptor ligand such as avidin, which is a noncovalently bound homotetrameric glycoprotein that binds to D-galactose receptor. D-galactose receptor is expressed on many cancer cells including ovarian, colon, gastric, and pancreatic cancer cells.
  • a targeting ligand also can be an antibody or antigen-binding fragment thereof that has binding affinity for a tumor specific antigen such as human epidermal growth factor receptor type 2 (HER2) expressed on the cell surface of some tumors. See, Ogawa et ah, 2009, supra.
  • an antibody or antigen-binding fragment thereof can be immobilized on a filter.
  • Such an antibody or antigen-binding fragment thereof can be immobilized on the filter before or after filtering the sample. It will be appreciated that immobilization of the antibody or fragment thereof on the filter, however, would not substantially contribute to the selection of target cells during the filtering process. Such an antibody or fragment thereof, however, can be useful for acting as a growth promoting ligand during the culture of the cells or after implantation in an immunodeficient non-human animal.
  • Antibody refers to a protein that generally comprises heavy chain polypeptides and light chain polypeptides. Antigen recognition and binding occurs within the variable regions of the heavy and light chains. Single domain antibodies having one heavy chain and one light chain and heavy chain antibodies devoid of light chains are also known. A given antibody comprises one of five types of heavy chains, called alpha, delta, epsilon, gamma and mu, the categorization of which is based on the amino acid sequence of the heavy chain constant region.
  • IgA immunoglobulin A
  • IgD immunoglobulin A
  • IgE immunoglobulin G
  • IgM immunoglobulin M
  • a given antibody also comprises one of two types of light chains, called kappa or lambda, the categorization of which is based on the amino acid sequence of the light chain constant domains.
  • IgG, IgD, and IgE antibodies generally contain two identical heavy chains and two identical light chains and two antigen combining domains, each composed of a heavy chain variable region (VH) and a light chain variable region (VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • IgA antibodies are composed of two monomers, each monomer composed of two heavy chains and two light chains (as for IgG, IgD, and IgE antibodies); in this way the IgA molecule has four antigen binding domains, each again composed of a VH and a VL.
  • Certain IgA antibodies are monomeric in that they are composed of two heavy chains and two light chains.
  • Secreted IgM antibodies are generally composed of five monomers, each monomer composed of two heavy chains and two light chains (as for IgG and IgE antibodies); in this way the IgM molecule has ten antigen binding domains, each again composed of a VH and a VL.
  • Antigen binding fragment of an antibody as the term is used herein refers to an antigen binding molecule that is not a complete antibody as defined above, but that still retains at least one antigen binding site. Antibody fragments often include a cleaved portion of a whole antibody, although the term is not limited to such cleaved fragments. Antigen binding fragments can include, for example, a Fab, F(ab')2, Fv, and single chain Fv (scFv) fragment.
  • An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived.
  • suitable antibodies or antigen binding fragments include linear antibodies, multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies (Poljak Structure 2(12): 1 121- 1 123 (1994); Hudson et al, J. Immunol. Methods 23(1-2): 177-189 (1994)), triabodies, tetrabodies), minibodies, chelating recombinant antibodies, intrabodies (Huston et al, Hum. Antibodies 10(3-4): 127-142 (2001); Wheeler et al. , Mol.
  • Non-limiting examples of antibodies or antigen-binding fragments thereof that act as growth promoting ligands include anti-CD3 antibodies for T cell tumors; anti-Ig antibodies for B cell tumors; or antibodies that can induce dimerization of class 1 growth factor receptors. See, for example, Fuh, et al. (1992) Science 256: 1677— 1680; Rui, et al. (1994) Endocrinology 135: 1299-1306; Schneider, et al. (1997) Blood 89: 473 ⁇ 182; Mahanta, et al. (2008) PLoS One. 3(4):e2054; and Spaargaren, et al. (1991) J Biol Chem. 266(3): 1733-9.
  • the filter can be implanted in a non-human animal, most commonly an immunodeficient non-human animal (e.g., an immunodeficient rodent such as an immunodeficient mouse or rat).
  • the immunodeficient non-human animal can be homozygous for the severe combined immune deficiency (SCID) spontaneous mutation (Prkdc scld ); homozygous for the nude spontaneous mutation (Foxnl nu/nu ); homozygous for a Ragl mutation;
  • the filter is surgically implanted into the immunodeficient animal subcutaneously.
  • the filter can be implanted under the neural crest, under the adrenal gland capsule, in the peritoneal cavity, or flank of the animal.
  • a compound such as a growth factor or reconstituted basement membrane matrix can be administered to the animal before, during, or after the filter is implanted.
  • one to four additional filters are implanted in the non- human animal, where each filter contains one or more target cells.
  • each filter contains one or more target cells.
  • one, two, three, or four filters can be implanted in the non-human animal.
  • Each filter can be obtained from a single filtration device or can be obtained from separate filtration devices.
  • all of the filters contain cells recovered from the same patient.
  • the filters can be implanted in different regions of the animal, e.g., in each flank or flank and abdomen of the animal.
  • the surface of the filter before implantation, can be contacted with a composition that can transition from a liquid to gel phase without lethal or toxic effects on the target cells, for example, without the use of chemicals or temperatures that would harm living cells, e.g., kill or inhibit the proliferative capacity of the cells.
  • the constituents of the compositions should not be toxic or lethal to cells or anti-proliferative.
  • the composition can be a hydrogel composed of crosslinked polymer chains, natural or synthetic in origin, such as PuramatrixTM (a synthetic peptide matrix) from 3 DM, Inc (Cambridge, MA), or the polyethylene (glycol) diacrylate-based, hyaluron based, or collagen based hydrogels from Glycosan BioSystems (Salt Lake City, UT).
  • PuramatrixTM a synthetic peptide matrix
  • 3 DM, Inc Conbridge, MA
  • polyethylene diacrylate-based, hyaluron based, or collagen based hydrogels from Glycosan BioSystems (Salt Lake City, UT).
  • Gels can be applied in liquid form to the filter and then transitioned to the gel phase by adding culture medium. Filters also can be contacted with MatrigelTM (BD Biosciences)
  • the composition also can contain one or more extracellular matrix components, e.g., proteoglycans (such as heparan sulfate, chondroitin sulfate, and keratan sulfate), hyaluronic acid, collagen type IV, elastin, fibronectin, and laminin. Growth factors or other molecules can be added to the compositions as needed for culture of the target cells.
  • proteoglycans such as heparan sulfate, chondroitin sulfate, and keratan sulfate
  • hyaluronic acid e.g., heparan sulfate, chondroitin sulfate, and keratan sulfate
  • hyaluronic acid e.g., heparan sulfate, chondroitin sulfate, and keratan sulfate
  • hyaluronic acid e.g., he
  • the filters can be stacked substantially on top of each other to produce a multi-layered three- dimensional culture device.
  • the surface of the filters can be contacted with the above-described composition that can transition from a liquid to gel phase without lethal or toxic effects on the target cells.
  • the surface of the filter can be contacted with a reconstituted basement membrane matrix.
  • the one or more filters before implanting, can be placed in a cell culturing device and cultured in the presence of a culture medium to, for example, assess viability or increase cell number.
  • the target cells once cell number of the target cells has increased, the target cells can be removed from the filter (e.g., by washing) and implanted in the immunodeficient animal.
  • the animal After implanting the one or more filters in the immunodeficient non-human animal, the animal can be monitored for growth of the cells or development of a tumor.
  • the implanted filter can include a substrate for a MMP or an in vivo activatable optical imaging probe as discussed above.
  • Development of a tumor in the animals confirms the presence of tumor cells in the fluid sample and is indicative of the metastasis potential of the cells.
  • a tumor can be removed from an animal and subjected to further in vitro or in vivo
  • a tumor or cells isolated from the tumor can be subjected to genomic, proteomic, immunocytochemical, or other molecular assays to further characterize the tumor and/or cells.
  • tissue specific and/or tumor specific reagents such as antibodies, probes, or PCR primers can be used to examine a tumor or cells isolated from the tumor.
  • responsiveness of the cells to one or more chemotherapeutic agents can be assessed by administering the chemotherapeutic of interest to the animal and monitoring responsiveness (e.g., by monitoring cell growth or cell death).
  • EXAMPLE 1 Use of the ScreenCell® Filtration Device
  • ScreenCell® ScreenCell® cell culture (CC)
  • the device is 19 cm long and includes a circular track-etched polycarbonate filter (e.g., from Whatman, EMD Millipore, Membrane Solution, or it4ip) with a smooth, flat and hydrophilic surface.
  • a circular track-etched polycarbonate filter e.g., from Whatman, EMD Millipore, Membrane Solution, or it4ip
  • the filter contains circular pores having a diameter of 6.5 ⁇ , randomly distributed throughout the filter (1 x 10 5 pores/cm 2 ).
  • RBCs red blood cells
  • 3 to 6 ml blood samples are diluted in 3 to 1 ml of ScreenCell® LC buffer, respectively. That is, a 3 ml blood sample is diluted with 3 ml of buffer while a 6 ml blood sample is diluted with 1 ml of buffer.
  • the sample is passed through the filtration device to which a vacuum tube is attached. Filtration is usually complete within approximately 2 minutes.
  • the nozzle/holder of the ScreenCell® device is undipped and removed from the filtration tank and the filter is released into a well of a 24-well tissue culture plate, by pushing down evenly a rod located at the bottom part of the filtration device. Adequate tissue culture medium and growth factors can be added into the well.
  • the filtration area of the ScreenCell® CC device filter is delimited by an O ring made of surgical inox with a numeric code to insure traceability of the filtered samples.
  • cells can be recovered from multiple portions of the same diluted blood sample. Each portion can be filtered through a different filtration device. When multiple filters are obtained, the filters can be laid successively upon each other as described above. For example, a filter can be released from a filtration device and placed on a 100 ⁇ layer of 1 : 1 Matrigel/Medium (M/M). Each successive filter is covered with 70 ⁇ of M/M before laying down the next filter.
  • M/M Matrigel/Medium
  • the last filter is covered with 70 ⁇ of M/M, and a sufficient volume of culture medium (e.g., culture medium containing fetal calf serum (FCS)) is added to cover the stack of filters to provide a three-dimensional culture device.
  • culture medium e.g., culture medium containing fetal calf serum (FCS)
  • FCS fetal calf serum
  • the sensitivity of the ScreenCell® device (described in Example 1) for isolating CTCs was assessed as follows. Twenty five independent experiments were conducted with fixed H2030 cells (an adenocarcinoma non-small cell lung cancer cell line from the American Type Culture Collection (ATCC); Catalog No. CRL-5914TM). The H2030 cells were cultured in flasks containing RPMI 1640 supplemented with 10% FCS and harvested by trypsinization. Cell viability was assessed by trypan blue exclusion. The cells were used in the experiments described below if viability was estimated to exceed 90%.
  • H2030 cells an adenocarcinoma non-small cell lung cancer cell line from the American Type Culture Collection (ATCC); Catalog No. CRL-5914TM.
  • the H2030 cells were cultured in flasks containing RPMI 1640 supplemented with 10% FCS and harvested by trypsinization. Cell viability was assessed by trypan blue exclusion. The cells were used in the experiments described below if viability was estimated
  • the H2030 cells were spiked into whole peripheral blood drawn from a healthy donor to yield a final concentration of 2 or 5 fixed H2030 cells per 1 mL of blood, and filtered through the ScreenCell® device as set forth in Example 1.
  • the average filtration time was 50 seconds. Cells on the filters were stained with hematoxylin and eosin, and counted.
  • the number of H2030 cells spiked into the blood sample versus the actual number of H2030 cells recovered in the sample is shown in Tables 1 and 2.
  • Tables 1 and 2 For the samples spiked with 5 cells, the average percentage of H2030 cells recovered was 91.2%, with an average of 4.56 ⁇ 0.71 cells recovered. See Table 1. In the samples spiked with 5 cells, no fewer than 3 cells were detected in all 25 samples.
  • the average percentage of H2030 cells recovered was 74% with an average of 1.480 ⁇ 0.71 cells recovered. See Table 2.
  • H2030 cells were harvested as indicated above, fixed, and pipetted directly into an Eppendorf tube containing filtration buffer. Cells were recovered using a Cytospin centrifuge and stained with hematoxylin and eosin. Under these conditions, the mean percentage of recovery was 82% for samples spiked with 2 cells and 88% for samples spiked with 5 cells. For the samples spiked with 2 cells, an average of 1.64 ⁇ 0.57 cells was recovered. For the samples spiked with 5 cells, an average of 4.40 ⁇ 0.71 cells was recovered.
  • the relative sensitivities of the ScreenCell® device versus direct cell collection were assessed through P-values calculated for unpaired unilateral Student test (0.19 and 0.20 for 2 and 5 spiked cells, respectively), unpaired bilateral Student test (0.39 and 0.41 for 2 and 5 cells respectively), and Fisher test (0.14 and 0.34 for 2 and 5 cells respectively). These tests showed that collection of 2 or 5 spiked tumor cells through the Screencell® device or by direct collection of the micropipetted cells directly into an Eppendorf tube resulted in similar sensitivities. Through the different series of tests using the Screencell® device and direct collection, similar numbers of cells were lost after 25 independent collections of 2 or 5 spiked tumor cells.
  • the percentage of cells lost through the Screencell® device was 26% (standard deviation (SD) was 0.71 with an average number of cells lost of 0.52), and 9% (SD was 0.65 with an average number of cell lost of 0.44) for 2 and 5 spiked H2030 cells respectively, while it was 18% (SD was 0.57 with an average number of cell lost of 0.36) and 12% (SD was 0.71 with an average number of cell lost of 0.60) through direct collection.
  • SD standard deviation
  • SD standard deviation
  • FIG. 8A is a photomicrograph of live H2030 cells following filtration through a ScreenCell® Cyto device. The mean percent recovery was 85% ⁇ 9%. The capacity of isolated H2030 cells to grow in tissue culture was further evaluated in eight independent experiments. In each experiment, isolated H2030 cells were able to grow and expand on the filter under adequate tissue culture conditions.
  • FIG. 8B is a photomicrograph of the cells from FIG. 8A after culturing of the filter for 4 days in culture medium.
  • human HT29 colorectal cancer cells were grown to 50% confluence and trypsinized to release the cells from the surface of the culture dish. The cells were washed in PBS and diluted to yield a final concentration of 10,000 cells per ⁇ . Normal human blood was collected in a sterile EDTA Vacutainer and used within 60 minutes of collection. Either 1000 or 10,000 HT29 cells were added to 6 ml of normal human blood and the blood was gently mixed. Six ml of blood containing the HT29 cells then was diluted with 1 ml of ScreenCell® LC dilution buffer and incubated for 2 minutes at room temperature.
  • Matrigel/Medium This solidified M/M pad was used to keep cells hydrated during the surgical procedure.
  • the filter was implanted under the skin of a Rag2 " " immunodeficient mouse and cell growth was monitored by palpation.
  • a tumor developed on both sides of the ScreenCell® filter within 3 weeks.
  • the mouse was sacrificed and the filter with attached tumor was removed and placed in a Petri dish. Cells from the tumor were cultured and stained with antibodies, confirming that the tumor was derived from HT29 cells.
  • Herrmann et al. (PLoS ONE, 2010, 5: 1-10) show that
  • CD44 high /CD24 high /EpCAM high HT29 cells selected in vivo had a cancer stem cell phenotype.

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Abstract

L'invention porte sur des procédés et des matériels pour augmenter le nombre de cellules cibles récupérées d'un échantillon fluide contenant des cellules. Les procédés comprennent l'isolation des cellules cibles sur un filtre, puis l'implantation du filtre contenant les cellules cibles dans un animal non humain immunodéficient, où au moins certaines des cellules cibles peuvent proliférer.
PCT/US2012/054241 2011-09-07 2012-09-07 Procédés d'augmentation du nombre de cellules cibles récupérées d'un échantillon fluide WO2013036819A1 (fr)

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CN201280054752.5A CN103917871A (zh) 2011-09-07 2012-09-07 增加从流体样品中所回收的靶细胞的数量的方法
CA2847891A CA2847891A1 (fr) 2011-09-07 2012-09-07 Procedes d'augmentation du nombre de cellules cibles recuperees d'un echantillon fluide
US14/342,888 US20150110717A1 (en) 2011-09-07 2012-09-07 Methods of increasing the number of target cells recovered from a fluid sample
EP12766511.5A EP2753926A1 (fr) 2011-09-07 2012-09-07 Procédés d'augmentation du nombre de cellules cibles récupérées d'un échantillon fluide
JP2014529908A JP2014526251A (ja) 2011-09-07 2012-09-07 流体サンプルから回収された標的細胞の数を増大させる方法

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WO2016094315A1 (fr) 2014-12-08 2016-06-16 Dana-Farber Cancer Institute, Inc. Appareil et procédé pour isoler des cellules cibles à partir d'un échantillon de fluide
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EP3561508A1 (fr) 2014-12-08 2019-10-30 Dana Farber Cancer Institute, Inc. Appareil et procédé permettant d'isoler des cellules cibles d'un échantillon de fluide

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